芳香族降解.docx

1、芳香族降解Preferential degradation of aromatic hydrocarbons in kerosene by a microbial consortiumHernando Bacosa*, Koichi Suto, Chihiro InoueGraduate School of Environmental Studies, Tohoku University, Aoba 6-6-20, Aramaki, Aoba-ku, Sendai, Miyagi 980-8579, JapanAbstract: Numerous studies on the biodegra

2、dation of petroleum products using total petroleum hydrocarbons (TPH) have been carried out; however, the biodegradation of equivalent carbon number (EC) based hydrocarbon fractions in mineral oil has attracted little attention. This study investigated the ability of a microbial consortium to degrad

3、e the EC fractions in kerosene, which was used as a representative mineral oil. Based on the cloning and sequencing of the 16S rRNA gene, the microbial community was predominantly identified as Betaproteobacteria of the genera Achromobacter, Alcaligenes, and Cupriavidus. Degradation experiments in s

4、ealed 120-ml vials containing 1% (w v_1) kerosene revealed that aromatic fractions were degraded faster than aliphatic fractions. Aromatic fractions EC 7e8 and EC 8e10 were completely degraded after three days while aliphatic fractions EC 6e8 and EC 8e10 were only partially degraded.The aromatic EC1

5、0e12 fractionwas the third most degraded, and the aliphatic EC10e12 and EC12e16 fractions were the least degraded fractions. The first-order rate constants for the aromatic fractions ranged from 0.12 d_1 to 0.51 d_1 and from 0.06 d_1 to 0.32 d_1 for the aliphatic fractions. The microbial consortium

6、preferentially utilized aromatic fractions, which are more toxic than aliphatic fractions. This finding is useful when considering risk-based bioremediation: A microbial community could potentially degrade the more Toxic aromatic hydrocarbon components in roleum-contaminated environments.1. Introduc

7、tion The contamination of soil,groundwater,and marine ecosystems with petroleum products as a result of human and industrial Activities is a widespread environmental problem. Petrochemical mixtures, such as mineral oil, may contain hundreds of aliphatic and aromatic hydrocarbon compounds with a wide

8、 range of properties and toxicities (Blomberg et al., 1997; Potter and Simmons, 1998). Aromatic hydrocarbons are more toxic and more persistent in the environment than are aliphatic compounds. The tremendous complexity of petroleum mixtures makes it difficult, if not impossible,to identify the indiv

9、idual components using currently available analytical methods. Thus, the complexity of the oil products and the lack of a toxicity reference make cleanup approaches and bioremediation strategies in contaminated sites an onerous task. The fractionation approach of the Total Petroleum Hydrocarbon Crit

10、eria Working Group (TPHCWG) provides a practical solution to these limitations. The TPHCWG has grouped petroleum hydrocarbons into aromatic and aliphatic groups, which are further categorized into 13 fractions based on their equivalent carbon number (EC) (Edwards et al., 1997; Gustafson et al., 1997

11、). The EC is related to the boiling point of the hydrocarbon compounds normalized to the boiling point of the n-alkanes or their retention time in a boiling point gas chromatography (GC) column. Compounds that belong in the same EC range are presumably similar in their environmental behaviors, degra

12、dability, and toxicity. The TPHCWG has assigned toxicity-based limit values to each of the EC fractions, which can be used to estimate the human non-carcinogenic health risks.The TPHCWG evaluated risk based on two criteriadthe reference dosage (RfD) and the reference concentration (RfC)dboth of whic

13、h terms were originally coined by the United States Environmental Protection Agency (USEPA). The RfD is “an estimate (with uncertainty spanning perhaps an order of magnitude) of daily exposure level for the human population, including sensitive subgroups, that is likely to be without appreciable ris

14、k of deleterious effects during a lifetime” (USEPA, 1989). The RfC is “an estimate (with uncertainty spanning perhaps an order of magnitude) of a continuous inhalation exposure to the human population, including sensitive subgroups, that is likely to be without an appreciable risk of deleterious eff

15、ects during a lifetime”(USEPA, 1994). Each fraction has a corresponding RfD and RfC that are used to evaluate the non-carcinogenic effects of exposure to that fraction (Vorhees et al., 1999). Furthermore, aromatic compounds, including benzene, toluene, ethylbenzene, and xylene(collectively known as

16、BTEX), as well as polycyclic aromatic hydrocarbons(PAHs), can pose a more serious risk to human health because of their carcinogenic and mutagenic potentials (Kstneret al., 1998; Lpez et al., 2008). Bioremediation is an attractive and environmental friendly approach for the cleanup of contaminated s

17、ites. It exploits the potential of naturally occurring microbial populations or, in some cases, introduces microorganisms with a known ability to degrade the contaminants. The application of a microbial consortium with a wide array of enzymatic abilities is more effective in the degradation of petro

18、leumusing a single isolate (Richard and Vogel, 1999;Rahman et al., 2002). Bioremediation of petroleum hydrocarboncontaminated sites has been the focus of many studies and the technique has been recently applied; these investigations used the total petroleum hydrocarbons (TPH) concentration to determ

19、ine the total mass of the contaminant in the environmental and as a measure of biodegradation (Al-Awadhi et al., 1996; Maila et al.,2005; Iturbe et al., 2007; Machackova et al., 2008). Similarly,numerous laboratory studies dealing with petroleum hydrocarbon biodegradation have also used the TPH conc

20、entration to measure the degradation of the contaminant (Hozumi et al., 2000; Okoh et al., 2001; Jamrah et al., 2007; Adams and Guzmn-Osorio,2008). However, this method does not consider the complexity of the pollutant and has neglected to consider that hydrocarbon components are associated with a r

21、isk to human health and the environment. Conventional cleanup or bioremediation approach that do not consider the risk to humans or the environment are costly and do not ensure success or the complete recovery of the contaminated site (Khan and Husain, 2001). As the TPH value does not provide inform

22、ation on the composition of the spilled oil,evaluating the efficiency of biodegradation based on EC fractions is a useful approach in risk-based and cost-effective bioremediation efforts. However, studies on the degradability of each compound and EC fraction in mineral oils are limited. Moreover, no

23、t much is known about the degradation of EC fractions in mineral oil in relation to microbial populations and specific environments. This study investigated the ability of a microbial consortium to degrade the aromatic and aliphatic EC fractions of kerosene, which was used as a representative minera

24、l oil, in liquid cultures.2. Materials and methods2.1. Chemicals and media Naphthalene, acenaphthylene, anhydrous sodium sulfate,dichloromethane, and n-hexane were obtained from Wako Pure Chemicals Industries (Osaka, Japan). ASTM D2887 aliphatic mix standard and silica gel grade 923 with 100e200 mes

25、h were procured from Sigma-Aldrich (Mo., USA). Toluene and ethylbenzene were purchased from GL Sciences (Tokyo, Japan). All chemicals were of the highest purity available, generally greater than 96% as listed by manufacturers. Kerosene was obtained from a local gas station. Bushnell Haas medium (BHM

26、) was used as the culture medium in this study. BHM is composed of the following: 1 g l_1 NH4NO3, 1 g l_1 K2HPO4,1 g l_1KH2PO4, 200mg l_1 MgSO4$7H2O, 50 mg l_1 FeCl3, and 20 mg l_1 CaCl2. The pH was adjusted to 7.0 with 1.0 M NaOH. The media was sterilized by autoclaving at 121 _C for 15 min. After

27、cooling,1 ml of filter-sterilized (0.2-mm filter) trace element solution was added. The trace element solution contained 3 mg l_1MnSO4$7H2O,3 mg l_1 ZnSO4$7H2O, 1 mg l_1 CoSO4$7H2O, and 1 mg l_1 (NH4)6Mo7O24$4H2O.2.2. Microbial consortium The microbial consortium was obtained through enrichment tech

28、niques from petroleum-contaminated soil at Yabase Oil Field in Akita Prefecture, Japan. Soil samples (10 g each) were added to 120-ml serum bottles containing 20 ml of BHM and 1% w v_1 kerosene.After two weeks at 30 _C in an orbital shaker, 1-ml aliquots were transferred to 19 ml of fresh BSM contai

29、ning 1% w v_1 kerosene as the sole carbon and energy source. To attain a stable kerosene degrading consortium, the culture was enriched through successive transfers every two weeks over six months.2.3. DNA extraction Bacterial cells were harvested from the kerosene-adapted microbial consortium by ce

30、ntrifugation at 10,000 rpm for 10 min in sterile plastic tubes. The supernatant was discarded and cell pellets were suspended in 40 ml of sterilized water. Freeze/thaw cycles for 15 min at_80 _C and 15min at roomtemperaturewere performed twice. Ten microliters of Proteinase K (1 mg ml_1) and 50 ml o

31、f TTNE buffer (40 mM TriseHCl, 1% Tween 20, 0.2% Nonidet P-40, 0.2 mM EDTA)were added for a total reaction volume of 100 ml. The proteinase K was activated by incubating the solution at 60 _C for 20min. TE saturated phenol (100 ml) was added before the sample was vortexed for 30 s.The sample was the

32、n centrifuged at 10,000 rpm for 10 min. The layer containing the nucleic acids was removed and purified by ethanol precipitation. DNA was dissolved in 1/10 diluted TE buffer and stored at _20 _C until use.2.4. PCR amplification Nearly full-length 16S rRNA gene fragments were amplified by PCR using t

33、he universal primers Eu 10F (5-AGAGTTTGATCCTGGCTCAG-3) corresponding to Escherichia coli positions 8e27 as a forward primer, and Eu 1500R (50-GGTTACCTTGTTACGACTT-30)corresponding to E. coli positions 1510e1492 as a reverse primer. PCR reactions were performed in a total volume of 50 ml containing 25 ml of PCR Master Mix (Promega, USA), 20 pmol o